Supplementary Figure 3: Construction and characterization of cre lentivirus that decreases the survival of infected newborn neurons in ALK5loxP/loxP mouse dentate gyrus.
From: ALK5-dependent TGF-β signaling is a major determinant of late-stage adult neurogenesis
(a) Design of the lentiviral vectors. (b) Immunohistochemical detection of eGFP labeled lentivirus-infected cells in the dentate gyrus of stereotaxically injected 2-month-old ALK5loxP/loxP mice. (c,d) Representative confocal images (c) and quantification (d) of lentivirus-injected dentate gyrus from 3-month-old ALK5loxP/loxP mice immunostained for p-Smad2 and eGFP. P-Smad2 signals were normalized to signals in uninfected cells in (d). Green or blue dashed circles highlight the cell bodies of eGFP+ or eGFP– neurons. n = 27, 21, 24, 23 neurons from three mice total for eGFP–/LV-eGFP, eGFP+/LV-eGFP, eGFP–/LV-cre-eGFP, eGFP+/LV-cre-eGFP group, respectively. (e) Representative orthogonal projections from confocal Z-stack images of LV-cre-eGFP lentivirus-injected dentate gyrus of2-month-old ALK5loxP/loxP mice immunostained for eGFP in combination with Sox2 plus GFAP, MCM2, Tbr2, DCX plus PCNA, and NeuN. DAPI stains nuclei. Scale bar is 100 μm in (b) and 10 μm in (c,e). (f) Quantification of the number of lentivirus-infected eGFP and DCX double-labeled newborn neurons in the dentate gyrus from LV-cre-eGFP and LV-eGFP stereotaxically injected ALK5loxP/loxP mice with 2 months of age at 6, 14, and 21 dpi. Cell numbers were normalized to values at 6 dpi. n = 4 mice per group, 4 sections per mouse. *P = 0.0355. Data are presented as mean + s.d. in (d) and mean + s.e.m. in (f). ****P < 0.0001. One-way ANOVA, Tukey's post-hoc test (d); one-tailed Student's t-test (f).
